The family of phosphoinositide 3-kinases (“PI3 kinase”, or “PI3K”) is ubiquitously expressed in cells, and their activation plays a major role in intracellular signal transduction. Activators of this enzyme include many cell surface receptors, especially those linked to tyrosine kinases. PI3K catalyzes the phosphorylation of membrane inositol lipids, with different family members producing different lipid products. Two of these products, phosphatidylinositol (3,4)-bisphosphate [PtdIns (3,4)P2] and phosphatidylinositol (3,4,5)-triphosphate [PtdIns (3,4,5)P3] act as “second messengers” that influence a variety of cellular processes and events.
PI3K was first identified as a heteromeric complex of two subunits: a 110 kDa cata-lytic subunit (p110α) and a 85 kDa regulatory subunit (p85α). Since then, eight additional PI3Ks have been identified: these are grouped into three main classes based on differences in their subunit structure and substrate preference in vitro. p110α falls into Class I, and is further categorized into Class Ia based on its mechanism of action in vivo. Two other close members in this group are p110β and p110δ. The p85 adapter subunit has two SH2 domains that allow PI3K to associate with cell surface receptors of the tyrosine kinase family, and are thereby critical to activate the enzyme, although a detailed mechanism of action is unknown.
Once PI3K is activated, it generates lipid products that act to stimulate many different cellular pathways. Many of these pathways have been described for the Class Ia group in a number of different cell types. It is evident that the cellular effects observed upon PI3K activation are the result of downstream targets of this enzyme. For example, protein kinase B (PKB) or AKT, and the related kinases protein kinases A and C (PKA and PKC) are activated by two phosphorylation events catalyzed by PDK1, an enzyme that is activated by PI3K.
A number of observations that link PI3K function with cell proliferation and inflammation point to a therapeutic role for PI3K inhibitors. In the area of oncology, results show that the p110α subunit of PI3K is amplified in ovarian tumors (L. Shayesteh et al., Nature Genetics (1999) 21:99-102). Further investigations have also shown that PI3K activity is elevated in ovarian cancer cell lines, and treatment with the known PI3K inhibitor LY 294002 decreases proliferation and increases apoptosis. These studies suggest that PI3K is an oncogene with an important role in ovarian cancer.
A malignant tumor of the central nervous system, glioblastoma, is highly resistant to radiation and chemotherapy treatments (S. A. Leibel et al., J Neurosurg (1987) 66:1-22). The PI3K signal transduction pathway inhibits apoptosis induced by cytokine withdrawal and the detachment of cells from the extracellular matrix (T. F. Franke et al., Cell (1997) 88:435-37). D. Haas-Kogan et al., Curr Biol (1998) 8:1195-98 have demonstrated that glioblastoma cells, in contrast to primary human astrocytes, have high PKB/AKT activity, and subsequently high levels of the lipid second messengers produced by PI3K activity. Addition of the known PI3K inhibitor LY 294002 reduced the levels of the lipid products and abolished the PKB/AKT activity in the glioblastoma cells. Additionally, evidence exists to support the misregulation of the PI 3-kinase-PKB pathway in these cells. The glioblastoma cells contain a mutant copy of the putative 3′ phospholipid phosphatase PTEN. This phosphatase normally removes the phosphate group from the lipid product, thus acting to regulate signaling through the PI3K pathways. When wild-type PTEN was expressed in the tumor cells PKB/AKT activity was abolished. These experiments suggest a role for PTEN in regulating the activity of the PI3K pathway in malignant human cells. In further work these investigators also observed that inhibition of PDK1 reduced PKB/AKT activity. PDK1, as described above, is a protein kinase activated by PI3K, and is likely responsible for inducing the events that lead to the activation of PKB/AKT activity. In addition, cell survival was dramatically reduced following treatment with antisense oligonucleotides against PDK1. Thus inhibitors of the PI3K pathway including PI 3-kinase, PDK1, and PKB/AKT are all potential targets for therapeutic intervention for glioblastoma.
Another potential area of therapeutic intervention for inhibitors of PI3K is juvenile myelomonocytic leukemia. The NF1 gene encodes the protein neurofibromin, a GTPase activating (“GAP”) protein for the small GTPase Ras. Immortalized immature myelomonocytic cells from NF1 −/− mice have been generated that have deregulated signaling through the Ras pathway, including the PI3K/PKB pathway. These cells undergo apoptosis when incubated with known inhibitors of PI3K, LY294002 and wortmannin, indicating a normal role for the protein in cell survival.
Wortmannin (G. Powis et al., Cancer Res (1994) 54:2419-23) was originally isolated from soil bacteria, while LY294002 (C. J. Vlahos et al., J Biol Chem (1994) 269:5241-48) is a derivative of the broad spectrum kinase inhibitor quercetin. The site of action of both inhibitors is the ATP-binding site. While wortmannin has an IC50 of 2-5 nM, the IC50 for LY294002 is 0.5-1.5 μM on purified PI3K. Extensive testing of these and other kinase inhibitors has shown that both are effective inhibitors of other unrelated enzymes with similar affinities (S.P. Davies et al., Biochem J (2000) 351:95-105). Further, neither inhibitor is isoform-specific on PI3K, and the therapeutic index is low, indicating that these two inhibitors have low pharmaceutical potential. However, the clear clinical significance of developing a selective PI3K inhibitor, both in oncology and inflammation, coupled with the inability of presently known inhibitors to show selectivity, indicates a pressing need for novel PI3K inhibitors.